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Objective Rotavirus A (RVA) remains the main causative agent of gastroenteritis in young children and the young of many mammalian and avian species. In this study we describe a RVA strain detected from a 6-month-old child from Central African Republic (CAR). Results We report the 11 open reading frame sequences of a G29-P[6]-I2-R2-C2-M2-A2-N2-T2-E2-H2 rotavirus strain, RVA/Human-wt/CAR/CAR91/2014/G29P[6]. Nine genes (VP1–VP3, VP6, NSP1–NSP5) shared 90–100% sequence similarities with genogroup 2 rotaviruses. Phylogenetically, backbone genes, except for VP3 and NSP4 genes, were linked with cognate gene sequences of human DS-1-like genogroup 2, hence their genetic origin. The VP3 and NSP4 genes, clustered genetically with both human and animal strains, an indication genetic reassortment human and animal RVA strains has taken place. The VP7 gene shared nucleotide (93–94%) and amino acid (95.5–96.7%) identities with Kenyan and Belgian human G29 strains, as well as to buffalo G29 strain from South Africa, while the VP4 gene most closely resembled P[6]-lineage I strains from Africa and Bangladesh (97%).

Background Following the implementation of rotavirus vaccination in 2006, severe acute gastroenteritis (AGE) due to group A rotavirus (RVA) has substantially declined in US children. We report the RVA genotype prevalence as well as coinfection data from 7 US New Vaccine Surveillance Network sites during 3 consecutive RVA seasons, 2014–2016 Methods A total of 1041 stool samples that tested positive for RVA by Rotaclone enzyme immunoassay were submitted to the Centers for Disease Control and Prevention (CDC) for RVA genotyping and multipathogen testing. Results A total of 795 (76%) samples contained detectable RVA when tested at the CDC. Rotavirus disease was highest in children < 3 years of age. Four G types (G1, G2, G9, and G12) accounted for 94.6% of strains while 2 P types (P[4] and P[8]) accounted for 94.7% of the strains. Overall, G12P[8] was the most common genotype detected in all 3 seasons. Stepwise conditional logistic analysis found year and study site were significant predictors of genotype. Twenty-four percent of RVA-positive specimens contained other AGE pathogens. Conclusions G12P[8] predominated over 3 seasons, but strain predominance varied by year and study site. Ongoing surveillance provides continuous tracking and monitoring of US genotypes during the postvaccine era.

The etiologic agent of an outbreak of pneumonia in Wuhan, China, was identified as severe acute respiratory syndrome coronavirus 2 in January 2020. A patient in the United States was given a diagnosis of infection with this virus by the state of Washington and the US Centers for Disease Control and Prevention on January 20, 2020. We isolated virus from nasopharyngeal and oropharyngeal specimens from this patient and characterized the viral sequence, replication properties, and cell culture tropism. We found that the virus replicates to high titer in Vero-CCL81 cells and Vero E6 cells in the absence of trypsin. We also deposited the virus into 2 virus repositories, making it broadly available to the public health and research communities. We hope that open access to this reagent will expedite development of medical countermeasures.

Background Acute gastroenteritis (AGE) burden, etiology, and severity in adults is not well characterized. We implemented a multisite AGE surveillance platform in 4 Veterans Affairs Medical Centers (Atlanta, Georgia; Bronx, New York; Houston, Texas; and Los Angeles, California), collectively serving >320 000 patients annually. Methods From 1 July 2016 to 30 June 2018, we actively identified inpatient AGE case patients and non-AGE inpatient controls through prospective screening of admitted patients and passively identified outpatients with AGE through stool samples submitted for clinical diagnostics. We abstracted medical charts and tested stool samples for 22 pathogens by means of multiplex gastrointestinal polymerase chain reaction panel followed by genotyping of norovirus- and rotavirus-positive samples. We determined pathogen-specific prevalence, incidence, and modified Vesikari severity scores. Results We enrolled 724 inpatients with AGE, 394 non-AGE inpatient controls, and 506 outpatients with AGE. Clostridioides difficile and norovirus were most frequently detected among inpatients (for AGE case patients vs controls: C. difficile, 18.8% vs 8.4%; norovirus, 5.1% vs 1.5%; P < .01 for both) and outpatients (norovirus, 10.7%; C. difficile, 10.5%). The incidence per 100 000 population was highest among outpatients (AGE, 2715; C. difficile, 285; norovirus, 291) and inpatients ≥65 years old (AGE, 459; C. difficile, 91; norovirus, 26). Clinical severity scores were highest for inpatient norovirus, rotavirus, and Shigella/enteroinvasive Escherichia coli cases. Overall, 12% of inpatients with AGE had intensive care unit stays, and 2% died; 3 deaths were associated with C. difficile and 1 with norovirus. C. difficile and norovirus were detected year-round with a fall/winter predominance. Conclusions C. difficile and norovirus were leading AGE pathogens in outpatient and hospitalized US veterans, resulting in severe disease. Clinicians should remain vigilant for bacterial and viral causes of AGE year-round.

Since 2013, group A rotavirus strains characterized as novel DS-1-like intergenogroup reassortant “equine-like G3” strains have emerged and spread across 5 continents among human populations in at least 14 countries. Here, we report a novel one-step TaqMan quantitative real-time reverse transcription-PCR assay developed to genotype and quantify the viral load for samples containing rotavirus equine-like G3 strains. Using a universal G forward primer and a newly designed reverse primer and TaqMan probe, we developed and validated an assay with a linear dynamic range of 227 to 2.3 × 10(9) copies per reaction and a limit of detection of 227 copies. The percent positive agreement, percent negative agreement, and precision of our assay were 100.00%, 99.63%, and 100.00%, respectively. This assay can simultaneously detect and quantify the viral load for samples containing DS-1-like intergenogroup reassortant equine-like G3 strains with high sensitivity and specificity, faster turnaround time, and decreased cost. It will be valuable for high-throughput screening of stool samples collected to monitor equine-like G3 strain prevalence and circulation among human populations throughout the world.

Background The introduction of rotavirus vaccine in the United States has reduced rotavirus disease burden, but outbreaks still occur. Complete-series rotavirus vaccination coverage is Methods Clinical history, signs and symptoms, and vaccination history were abstracted for the 26 patients residing in the facility during the time of the outbreak. A case-patient was defined as one who experienced 3 or more loose stools in a period of 24 hours with onset between April 17 and May 17, 2017. Stool samples from 14 resident patients were tested for rotavirus with reverse-transcription polymerase chain reaction. Results The median patient age at the facility was 2.9 years. Of the 26 resident patients, 22 (85%) met the case definition. One child died. Stool samples from 11 case-patients were positive according to reverse-transcription polymerase chain reaction for rotavirus. Fifteen case-patients were unvaccinated against rotavirus; 3 were partially vaccinated, and 2 were fully vaccinated. Vaccination status could not be completely determined in 2 cases. Conclusions An outbreak of rotavirus affected nearly all resident patients of a subacute care facility and caused 1 death. Because of recommendations against giving rotavirus vaccine in an intensive care setting, infants who require a prolonged intensive care stay might age out of rotavirus vaccine eligibility (the first dose must be given before 15 weeks of age according to Advisory Committee on Immunization Practices recommendations). The result is a vulnerable population of unvaccinated infants who might later congregate in another care setting.

For over a decade, the New Vaccine Surveillance Network (NVSN) has conducted active rotavirus (RVA) strain surveillance in the USA. The evolution of RVA in the post-vaccine introduction era and the possible effects of vaccine pressure on contemporary circulating strains in the USA are still under investigation. Here, we report the whole-gene characterization (eleven ORFs) for 157 RVA strains collected at seven NVSN sites during the 2014 through 2016 seasons. The sequenced strains included 52 G1P[8], 47 G12P[8], 18 G9P[8], 24 G2P[4], 5 G3P[6], as well as 7 vaccine strains, a single mixed strain (G9G12P[8]), and 3 less common strains. The majority of the single and mixed strains possessed a Wa-like backbone with consensus genotype constellation of G1/G3/G9/G12-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1, while the G2P[4], G3P[6], and G2P[8] strains displayed a DS-1-like genetic backbone with consensus constellation of G2/G3-P[4]/P[6]/P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2. Two intergenogroup reassortant G1P[8] strains were detected that appear to be progenies of reassortment events between Wa-like G1P[8] and DS-1-like G2P[4] strains. Two Rotarix® vaccine (RV1) and two RV5 derived (vd) reassortant strains were detected. Phylogenetic and similarity matrices analysis revealed 2–11 sub-genotypic allelic clusters among the genes of Wa- and DS-1-like strains. Most study strains clustered into previously defined alleles. Amino acid (AA) substitutions occurring in the neutralization epitopes of the VP7 and VP4 proteins characterized in this study were mostly neutral in nature, suggesting that these RVA proteins were possibly under strong negative or purifying selection in order to maintain competent and actual functionality, but fourteen radical (AA changes that occur between groups) AA substitutions were noted that may allow RVA strains to gain a selective advantage through immune escape. The tracking of RVA strains at the sub-genotypic allele constellation level will enhance our understanding of RVA evolution under vaccine pressure, help identify possible mechanisms of immune escape, and provide valuable information for formulation of future RVA vaccines.

Background Despite the global roll-out of rotavirus vaccines (RotaTeq/Rotarix / ROTAVAC/Rotasiil), mortality and morbidity due to group A rotavirus (RVA) remains high in sub-Saharan Africa, causing 104,000 deaths and 600,000 hospitalizations yearly. In Cameroon, Rotarix™ was introduced in March 2014, but, routine laboratory diagnosis of rotavirus infection is not yet a common practice, and vaccine effectiveness studies to determine the impact of vaccine introduction have not been done. Thus, studies examining RVA prevalence post vaccine introduction are needed. The study aim was to determine RVA prevalence in severe diarrhoea cases in Littoral region, Cameroon and investigate the role of other diarrheagenic pathogens in RVA-positive cases. Methods We carried out a study among hospitalized children Results The ELISA assay detected RVA antigen in 54.6% (71/130) of specimens, with 45, positive by VP6 RT-PCR and 54, positive using Luminex xTAG GPP. Luminex GPP was able to detect all 45 VP6 RT-PCR positive samples. Co-infections were found in 63.0% (34/54) of Luminex positive RVA infections, with Shigella (35.3%; 12/34) and ETEC (29.4%; 10/34) detected frequently. Of the 71 ELISA positive RVA cases, 57.8% (41/71) were fully vaccinated, receiving two doses of Rotarix. Conclusion This study provides insight on RVA prevalence in Cameroon, which could be useful for post-vaccine epidemiological studies, highlights higher than expected RVA prevalence in vaccinated children hospitalized for diarrhoea and provides the trend of RVA co-infection with other enteric pathogens. RVA genotyping is needed to determine circulating rotavirus genotypes in Cameroon, including those causing disease in vaccinated children.

The etiologic agent of the outbreak of pneumonia in Wuhan China was identified as severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) in January, 2020. The first US patient was diagnosed by the State of Washington and the US Centers for Disease Control and Prevention on January 20, 2020. We isolated virus from nasopharyngeal and oropharyngeal specimens, and characterized the viral sequence, replication properties, and cell culture tropism. We found that the virus replicates to high titer in Vero-CCL81 cells and Vero E6 cells in the absence of trypsin. We also deposited the virus into two virus repositories, making it broadly available to the public health and research communities. We hope that open access to this important reagent will expedite development of medical countermeasures.Article SummaryScientists have isolated virus from the first US COVID-19 patient. The isolation and reagents described here will serve as the US reference strain used in research, drug discovery and vaccine testing.

Group A rotavirus (RVA) associated diarrhoea in piglets represents one of the major causes of morbidity and mortality in pig farms worldwide. A diarrhoea outbreak occurred among nomadic piglets in north-western district of Bangladesh in February 2014. Outbreak investigation was performed to identify the cause, epidemiologic and clinical features of the outbreak. Rectal swabs and clinical information were collected from diarrhoeic piglets (n = 36). Rectal swabs were tested for RVA RNA by real-time reverse transcription polymerase chain reaction (rRT-PCR) using NSP3-specific primers. The G (VP7) and P (VP4) genes were typed by conventional RT-PCR and sanger sequencing and full genome sequences were determined using next-generation sequencing. We found the attack rate was 61% (50/82) among piglets in the nomadic pig herd, and the case fatality rate was 20% (10/50) among piglets with diarrhoea. All study piglets cases had watery diarrhoea, lack of appetite or reluctance to move. A novel RVA strain with a new P[49] genotype combined with G4 was identified among all piglets with diarrhoea. The genome constellation of the novel RVA strains was determined to be G4-P[49]-I1-R1-C1-M1-A8-N1-T7-E1-H1. Genetic analysis shows that the novel G4P[49] strain is similar to Indian and Chinese porcine or porcine-like G4 human strains and is genetically distant from Bangladeshi human G4 strains. Identification of this novel RVA strain warrants further exploration for disease severity and zoonotic potential.

Group A Rotaviruses (RVAs) are the most important etiological agents of acute gastroenteritis (AGE) in children less than 5 years of age. Mortality resulting from RVA gastroenteritis is higher in developing countries than in developed ones, causing a huge public health burden in global regions like Africa and South-East Asia. This study reports RVA genotypes detected in Ashaiman, Greater Accra Region, Ghana, in the postvaccine introduction era for the period 2014-2016. Stool samples were collected from children less than 5 years of age who visited Ashaiman Polyclinic with AGE from November 2014 to May 2015 and from December 2015 to June 2016. The samples were tested by enzyme immunoassay (EIA), and one-step multiplex reverse transcription polymerase chain reaction was performed on the EIA positive samples for gel-based binomial genotyping. Of the 369 stool samples collected from children with AGE, 145 (39%) tested positive by EIA. Five VP7 (G1, G3, G9, G10, and G12) and three VP4 (P[4], P[6] and P[8]) genotypes were detected. Eight G/P combinations were identified of which, G3P[6], G12P[8], G1P[8], and G9P[4] were the most prevalent and responsible for 93 (68%) of the AGE cases, and seven mixed-types were detected which represented 8% of the RVA cases. High prevalence, diversity, and mixed-types of RVAs were detected from Ashaiman with the emergence of unusual genotypes.

Background Acute gastroenteritis (AGE) in hematopoietic cell transplant (HCT) patients causes significant morbidity and mortality. Data regarding the longitudinal assessment of infectious pathogens during symptomatic AGE and asymptomatic periods, particularly in children, are limited. We investigated the prevalence of AGE-associated infectious pathogens in children undergoing allogeneic HCT. Methods From March 2015 through May 2016, 31 pediatric patients at 4 US children’s hospitals were enrolled and had stool collected weekly from pre-HCT through 100 days post-HCT for infectious AGE pathogens by molecular testing. Demographics, clinical symptoms, antimicrobials, vaccination history, and outcomes were manually abstracted from the medical record into a standardized case report form. Results We identified a pathogen in 18% (38/206) of samples, with many detections occurring during asymptomatic periods. Clostridioides difficile was the most commonly detected pathogen in 39% (15/38) of positive specimens, although only 20% (3/15) of C. difficile–positive specimens were obtained from children with diarrhea. Detection of sapovirus, in 21% (8/38) of pathogen-positive specimens, was commonly associated with AGE, with 87.5% of specimens obtained during symptomatic periods. Norovirus was not detected, and rotavirus was detected infrequently. Prolonged shedding of infectious pathogens was rare. Conclusions This multicenter, prospective, longitudinal study suggests that the epidemiology of AGE pathogens identified from allogeneic HCT patients may be changing. Previously reported viruses, such as rotavirus and norovirus, may be less common due to widespread vaccination and institution of infection control precautions, and emerging viruses such as sapoviruses may be increasingly recognized due to the use of molecular diagnostics.

Before the introduction of rotavirus vaccine in 2006, rotavirus was the most common cause of severe diarrhea among U.S. children (1). Currently, two rotavirus vaccines are licensed for use in the United States, both of which have demonstrated good field effectiveness (78%-89%) against moderate to severe rotavirus illness (2), and the use of these vaccines has substantially reduced the prevalence of rotavirus in the United States (3). However, the most recent national vaccine coverage estimates indicate lower full rotavirus vaccine-series completion (73%) compared with receipt of at least 3 doses of vaccines containing diphtheria, tetanus, and pertussis antigens (95%), given on a similar schedule to rotavirus vaccines (4). In the postvaccine era in the United States, rotavirus activity persists in a biennial pattern (3). This report describes three rotavirus outbreaks that occurred in California in 2017. One death was reported; however, the majority of cases were associated with mild to moderate illness, and illness occurred across the age spectrum as well as among vaccinated children. Rotavirus vaccines are designed to mimic the protective effects of natural infection and are most effective against severe rotavirus illness (2). Even in populations with high vaccination coverage, some rotavirus infections and mild to moderate illnesses will occur. Rotavirus vaccination should continue to be emphasized as the best means of reducing disease prevalence in the United States.

Group A rotaviruses are the major cause of severe gastroenteritis in the young of mammals and birds. This report describes characterization of an unusual G20P[28] rotavirus strain detected in a 24 month old child from Suriname. Genomic sequence analyses revealed that the genotype constellation of the Suriname strain RVA/Human-wt/SUR/2014735512/2013/G20P[28] was G20-P[28]-I13-R13-C13-M12-A23-N13-T15-E20-H15. Genes VP1, VP2, VP3, NSP1, NSP2, NSP3, NSP4 and NSP5 were recently assigned novel genotypes by the Rotavirus Classification Working Group (RCWG). Three of the 11 gene segments (VP7, VP4, VP6) were similar to cognate gene sequences of bat-like human rotavirus strain Ecu534 from Ecuador and the VP7, NSP3 and NSP5 gene segments of strain RVA/Human-wt/SUR/2014735512/2013/G20P[28] were found to be closely related to gene sequences of bat rotavirus strain 3081/BRA detected in Brazil. Although distantly related, the VP1 gene of the study strain and bat strain BatLi09 detected in Cameroon in 2014 are monophyletic. The NSP1 gene was found to be most closely related to human strain QUI-35-F5 from Brazil. These findings suggest that strain RVA/Human-wt/SUR/2014735512/2013/G20P[28] represents a zoonotic infection from a bat host.

Background Histo-blood group antigens (HBGAs), whose expression is controlled in part by fucosyltransferase 2 (FUT2) and 3 (FUT3) genes, serve as receptors for norovirus and rotavirus. Individuals without functional FUT2 (nonsecretors) or FUT3 (Lewis-negative) genes may have decreased susceptibility to norovirus and rotavirus infections. As the prevalence of secretor and Lewis status can vary by race and ethnicity, we assessed this association in a US Veteran population. Methods Stool and saliva specimens were collected from acute gastroenteritis (AGE) cases and age- and time-matched controls through a multisite, active surveillance platform at four Veterans Affairs hospitals (Atlanta, Bronx, Houston, Los Angeles). Stool specimens were tested with the FilmArray Gastrointestinal Panel; norovirus and rotavirus positive specimens were genotyped. Saliva specimens were analyzed for HBGA expression by EIA using glycan-specific monoclonal antibodies and lectins. Chi-squared and Fisher’s exact tests were conducted to evaluate associations between secretor and Lewis status and infection with norovirus or rotavirus. Results From November 4, 2015–December 30, 2017, 670 AGE cases and 319 controls provided both stool and saliva specimens. Norovirus (21 GII.4 Sydney, 13 GII non-4, 7 GI, 10 untyped) and rotavirus (13 G12P[8], 1 G2P[4], 1 untyped) positive cases were more likely to be secretor positive (90% and 100%, respectively) compared with controls (76%) (P = 0.03 for both). Infections with GII.4 Sydney norovirus (P < 0.01) and G12P[8] rotavirus (P < 0.05) were significantly associated with secretor status. This association was not observed with other norovirus or rotavirus genotypes. No association was observed between Lewis status, race, or ethnicity and infection with norovirus or rotavirus. Conclusion Norovirus and rotavirus infections among a US Veteran population were associated with secretor status in a genotype-dependent manner, and with GII.4 Sydney norovirus and G12P[8] rotavirus, the most common strains. These associations are consistent with previously reported results, and suggest that the efficacy of interventions, such as vaccines, should include consideration of secretor status and predominantly circulating virus strains. Disclosures R. L. Atmar, Takeda Vaccines, Inc.: Investigator, Research grant. V. C. Marconi, ViiV: Investigator, Research support and Salary. Gilead: Investigator, Research support. Bayer: Investigator, Research support.

The current two-step VP7 and VP4 genotyping RT-PCR assays for rotaviruses have been linked consistently to genotyping failure in an estimated 30% of RVA positive samples worldwide. We have developed a VP7 and VP4 multiplexed one-step genotyping assays using updated primers generated from contemporary VP7 and VP4 sequences. To determine assay specificity and sensitivity, 17 reference virus strains, 6 non-target gastroenteritis viruses and 725 clinical samples carrying the most common VP7 (G1, G2, G3, G4, G9, and G12) and VP4 (P[4], P[6], P[8], P[9] and P[10]) genotypes were tested in this study. All reference RVA strain targets yielded amplicons of the expected sizes and non-target genotypes and gastroenteritis viruses were not detected by either assay. Out of the 725 clinical samples tested, the VP7 and VP4 assays were able to assigned specific genotypes to 711 (98.1%) and 714 (98.5%), respectively. The remaining unassigned samples were re-tested for RVA antigen using EIA and qRT-PCR assays and all were found to be negative. The overall specificity, sensitivity and limit of detection of the VP7 assay were in the ranges of 99.0–100%, 94.0–100% and 8.6 × 101 to 8.6 × 102 copies of RNA/reaction, respectively. For the VP4 assay, the overall specificity, sensitivity and limit of detection assay were in the ranges of 100%, 94.0–100% and ≤1 to 8.6 × 102 copies of RNA/reaction, respectively. Here we report two highly robust, accurate, efficient, affordable and documentable gel-based genotyping systems which are capable of genotyping 97.8% of the six common VP7 and 98.3% of the five common VP4 genotypes of RVA strains which are responsible for approximately 88.2% of all RVA infections worldwide.

Rotavirus genotyping is useful for surveillance purposes especially in areas where rotavirus vaccination has been or will be implemented. RT-PCR based molecular methods have been applied widely, but quantitative assays targeting a broad spectrum of genotypes have not been developed. Three real time RT-PCR panels were designed to identify G1, G2, G9, G12 (panel GI), G3, G4, G8, G10 (panel GII), and P[4], P[6], P[8], P[10], P[11] (panel P), respectively. An assay targeting NSP3 was included in both G panels as an internal control. The cognate assays were also formulated as one RT-PCR-Luminex panel for simultaneous detection of all the genotypes listed above plus P[9]. The assays were evaluated with various rotavirus isolates and 89 clinical samples from Virginia, Bangladesh and Tanzania, and exhibited 95% (81/85) sensitivity compared with the conventional RT-PCR-Gel-electrophoresis method, and 100% concordance with sequencing. Real time assays identified a significantly higher rate of mixed genotypes in Bangladeshi samples than the conventional gel-electrophoresis-based RT-PCR assay (32.5% versus 12.5%, P0.05). In these mixed infections, the relative abundance of the rotavirus types could be estimated by Cq values. These typing assays detect and discriminate a broad range of G/P types circulating in different geographic regions with high sensitivity and specificity and can be used for rotavirus surveillance.

Background The severity of acute gastroenteritis (AGE) in adult populations and the relative contribution of specific pathogens is not well characterized. In 2016, we implemented a multisite AGE surveillance platform in 4 VA hospitals (Atlanta, Bronx, Houston and Los Angeles), collectively serving > 320,000 patients annually. Methods Inpatient AGE cases and age- and time-matched non-AGE controls were identified through prospective screening of admissions using standardized case definitions. Stool samples were tested for 22 pathogens using the FilmArray® Gastrointestinal Panel. Medical conditions were analyzed as risk factors for AGE by multivariate logistic regression. Results From July 2016 to June 2018, 731 cases and 399 controls were enrolled. Risk factors for AGE cases included HIV-positive status (adjusted odds ratio [aOR] 4.6; 95% confidence interval [CI] 1.6–12.9; P < 0.01), severe kidney disease (aOR 4.5; 95% CI 2.0–9.8; P < 0.01), and immunosuppressive therapy (aOR 4.0; 95% CI 1.2–13.3]; P = 0.02). Clostridioides difficile and norovirus were the most commonly detected pathogens in cases (18% and 5%, respectively); detection of these pathogens in cases was significantly higher than detection in controls (8% and 2%, respectively; P < 0.01 for both). The median duration of hospital stay was longer for C. difficile compared with norovirus cases (5 vs. 3 days; P < 0.01), and cases with both pathogens had intensive care unit (ICU) stays (C. difficile: 18%; norovirus: 8%; P = 0.2). Fourteen deaths occurred among AGE cases; 2 were associated with C. difficile and 1 with norovirus; the remainder did not have a clear etiology or pathogen detected. C. difficile and norovirus were detected year-round with a fall and winter predominance; C. difficile prevalence was highest in October, while norovirus prevalence was six times higher in December than in summer months. Conclusion This surveillance platform captured cases of severe AGE, including ICU stays and deaths, among hospitalized US Veterans. C. difficile and norovirus were leading pathogens in AGE cases. These findings can help guide appropriate clinical management of AGE patients and inform public health efforts to quantify and address the associated burden of disease through targeted interventions. Disclosures All authors: No reported disclosures.

Background Viruses are frequently implicated in acute gastroenteritis (AGE) outbreaks, yet the endemic burden of norovirus and rotavirus disease in adult populations is not well characterized. In 2016, we implemented a multisite AGE surveillance platform capturing cases and controls in 4 VA hospitals (Atlanta, Bronx, Houston, and Los Angeles), collectively serving >320,000 patients annually. Methods Inpatient AGE cases and age- and time-matched controls were identified through prospective screening of admissions via standardized case definitions. Outpatient cases were passively identified using stool samples submitted for routine clinical microbiological diagnostics. Samples were tested with the FilmArray Gastrointestinal Panel, followed by genotyping of virus positives. Incidence was estimated using population denominators of unique patients served annually by site. Results From December 1, 2016 to February 28, 2018, 875 cases (496 inpatients, 379 outpatients), and 374 controls were enrolled. Norovirus and rotavirus prevalence was highest among outpatient AGE cases (11.6% and 2.9%, respectively) followed by inpatient cases (3.4% and 1.6%, respectively); few controls were positive (norovirus, 1.3%; rotavirus, 0%). Norovirus-associated inpatient incidence was 15.2 per 100,000 population (range by site: 10.7–19.9/100,000) and rotavirus-associated inpatient incidence was 7.5 per 100,000 population (range by site: 0–12.8/100,000). The predominant norovirus genotype was GII.P16-GII.4 Sydney (50%), and rotavirus genotype was G12P[8] (83%). Norovirus was detected every calendar month and peaked in December–January, while rotavirus peaked in April. Nine deaths were documented among AGE inpatient cases, including one norovirus-associated death. Conclusion Implementation of a multisite AGE surveillance platform captured a wide spectrum of illness for norovirus and rotavirus in US Veterans including outpatient visits, inpatient hospitalizations, and one norovirus-associated death. Norovirus was the leading viral pathogen and was detected year-round. Ongoing surveillance using this platform will allow for further characterization of the pathogen distribution and associated AGE disease burden in adults. Disclosures V. Marconi, ViiV: Investigator, Research support and Salary. Bayer: Investigator, Research support. Gilead: Investigator, Research support.

Group A rotaviruses (RV-A) are the leading cause of viral gastroenteritis in children worldwide and genotype G9P[8] is one of the five most common genotypes detected in humans. In order to gain insight into the degree of genetic variability of G9P[8] strains circulating in Cameroon, stool samples were collected during the 1999–2000 rotavirus season in two different geographic regions in Cameroon (Southwest and Western Regions). By RT-PCR, 15 G9P[8] strains (15/89 = 16.8%) were identified whose genomic configurations was subsequently determined by complete or partial gene sequencing. In general, all Cameroonian G9 strains clustered into current globally-spread sublineages of the VP7 gene and displayed 86.6–100% nucleotide identity amongst themselves and 81.2–99.5% nucleotide identity with global G9 strains. The full genome classification of all Cameroonian strains was G9-P[8]-I1–R1–C1–M1–A1–N1–T1–E1–H1 but phylogenetic analysis of each gene revealed that the strains were spread across 4 or more distinct lineages. An unusual strain, RVA/Human-wt/CMR/6788/1999/G9P[8], which shared the genomic constellation of other Cameroonian G9P[8] strains, contained a novel G9 subtype which diverged significantly (18.8% nucleotide and 19% amino acid distance) from previously described G9 strains. Nucleotide and amino acid alignments revealed that the 3′ end of this gene is highly divergent from other G9 VP7 genes suggesting that it arose through extensive accumulation of point mutations. The results of this study demonstrate that diverse G9 strains circulated in Cameroon during 1999–2000.

Background Group A rotaviruses (RVA) are a significant cause of pediatric gastroenteritis worldwide. The New Vaccine Surveillance Network (NVSN) has conducted active surveillance for RVA at pediatric hospitals and emergency departments at 3-7 geographically diverse sites in the United States since 2006. Methods Over 6 consecutive years, from 2008 to 2013, 1523 samples from NVSN sites that were tested positive by a Rotaclone enzyme immunoassay were submitted to the Centers for Disease Control and Prevention for genotyping. Results In the 2009, 2010, and 2011 seasons, genotype G3P[8] was the predominant genotype throughout the network, with a 46%-84% prevalence. In the 2012 season, G12P[8] replaced G3P[8] as the most common genotype, with a 70% prevalence, and this trend persisted in 2013 (68.0% prevalence). Vaccine (RotaTeq; Rotarix) strains were detected in 0.6%-3.4% of genotyped samples each season. Uncommon and unusual strains (eg, G8P[4], G3P[24], G2P[8], G3P[4], G3P[6], G24P[14], G4P[6], and G9P[4]) were detected sporadically over the study period. Year, study site, and race were found to be significant predictors of genotype. Conclusions Continued active surveillance is needed to monitor RVA genotypes in the United States and to detect potential changes since vaccine licensure.

Background.Group A rotavirus (RVA) infection is the major cause of acute gastroenteritis (AGE) in young children worldwide. Introduction of two live-attenuated rotavirus vaccines, RotaTeq® and Rotarix®, has dramatically reduced RVA associated AGE and mortality in developed as well as in many developing countries. High-throughput methods are needed to genotype rotavirus wild-type strains and to identify vaccine strains in stool samples. Quantitative RT-PCR assays (qRT-PCR) offer several advantages including increased sensitivity, higher throughput, and faster turnaround time.Methods.In this study, a one-step multiplex qRT-PCR assay was developed to detect and genotype wild-type strains and vaccine (Rotarix® and RotaTeq®) rotavirus strains along with an internal processing control (Xeno or MS2 RNA). Real-time RT-PCR assays were designed for VP7 (G1, G2, G3, G4, G9, G12) and VP4 (P[4], P[6] and P[8]) genotypes. The multiplex qRT-PCR assay also included previously published NSP3 qRT-PCR for rotavirus detection and Rotarix® NSP2 and RotaTeq® VP6 qRT-PCRs for detection of Rotarix® and RotaTeq® vaccine strains respectively. The multiplex qRT-PCR assay was validated using 853 sequence confirmed stool samples and 24 lab cultured strains of different rotavirus genotypes. By using thermostablerTthpolymerase enzyme, dsRNA denaturation, reverse transcription (RT) and amplification (PCR) steps were performed in single tube by uninterrupted thermocycling profile to reduce chances of sample cross contamination and for rapid generation of results. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments.Results.The VP7 qRT-PCRs exhibited 98.8–100% sensitivity, 99.7–100% specificity, 85–95% efficiency and a limit of detection of 4–60 copies per singleplex reaction. The VP7 qRT-PCRs exhibited 81–92% efficiency and limit of detection of 150–600 copies in multiplex reactions. The VP4 qRT-PCRs exhibited 98.8–100% sensitivity, 100% specificity, 86–89% efficiency and a limit of detection of 12–400 copies per singleplex reactions. The VP4 qRT-PCRs exhibited 82–90% efficiency and limit of detection of 120–4000 copies in multiplex reaction.Discussion.The one-step multiplex qRT-PCR assay will facilitate high-throughput rotavirus genotype characterization for monitoring circulating rotavirus wild-type strains causing rotavirus infections, determining the frequency of Rotarix® and RotaTeq® vaccine strains and vaccine-derived reassortants associated with AGE, and help to identify novel rotavirus strains derived by reassortment between vaccine and wild-type strains.

Summary Objectives To assess the burden of rotavirus disease in Guatemala, in view of the recent introduction of a national rotavirus vaccination programme. Methods We examined data from an active, facility-based surveillance system in Santa Rosa, Guatemala, from October 2007 through September 2009 among children

We report the genomic characterization of a rare human G8P[14] rotavirus strain, identified in a stool sample from Guatemala (GTM) during routine rotavirus surveillance. This strain was designated as RV A/Human-wt/GTM/2009726790/2009/G8P[14], with a genomic constellation of G8-P[14]-I2-R2-C2-M2-A13-N2-T6-E2-H3. The VP4 gene occupied lineage VII within the P[14] genotype. Phylogenetic analysis of each genome segment revealed close relatedness to several zoonotic simian, guanaco and bovine strains. Our findings suggest that strain RVA/Human-wt/GTM/2009726790/2009/G8P[14] is an example of a direct zoonotic transmission event. The results of this study reinforce the potential role of interspecies transmission and reassortment in generating novel and rare rotavirus strains which infect humans.

Background Rotaviruses are the major cause of severe dehydrating diarrhea in children throughout the world. Enzyme immunoassays (EIAs) have been the standard method for detection of rotavirus in stool specimens since the 1980s. The World Health Organization (WHO) Rotavirus Surveillance Network has proposed including three EIA kits in the WHO-GSM (Global Management System/Systeme Mondial de Gestion) catalog for easy procurement of EIA kits by participating rotavirus surveillance network laboratories. Objectives In this study, we conducted a comparative analysis of 3 commercially available enzyme immunoassay kits: Premier™ Rotaclone® (Meridian Bioscience, Inc.), ProSpecT™ (Oxoid, Ltd.) and RIDASCREEN® (R-biopharm AG) for rotavirus diagnostics. Study design Using reverse-transcriptase-PCR (RT-PCR) as the gold standard, the 3 EIA kits were evaluated by testing a stool panel consisting of 56 rotavirus-positive and 54 rotavirus negative samples. Results The sensitivities of the Premier™ Rotaclone®, ProSpecT™ and RIDASCREEN® kits were 76.8%, 75% and 82.1%, respectively, but did not differ significantly. The specificity of all the 3 kits was 100%. The use of RT-PCR as a gold standard lowered the observed sensitivity of all 3 EIA kits but helps to reduce equivocal results that can be seen when another EIA or other non-molecular methods are used as the reference assay in comparison studies. Conclusion Our study found that all three kits are suitable for use by rotavirus surveillance programs.

In the mouse, more than 16 loci are associated with mutant phenotypes that include defective pigmentation, aberrant targeting of lysosomal enzymes, prolonged bleeding, and immunodeficiency, the result of defective biogenesis of cytoplasmic organelles: melanosomes, lysosomes, and various storage granules. Many of these mouse mutants are homologous to the human Hermansky–Pudlak syndrome (HPS), Chediak–Higashi syndrome, and Griscelli syndrome. We have mapped and positionally cloned one of these mouse loci, buff ( bf ), which has a mutant phenotype similar to that of human HPS. Mouse bf results from a mutation in Vps33a and thus is homologous to the yeast vacuolar protein-sorting mutant vps33 and Drosophila carnation ( car ). This is the first found defect of the class C vacuole/prevacuole-associated target soluble N -ethylmaleimide-sensitive factor attachment protein receptor (t-SNARE) complex in mammals and the first mammalian mutant found that is directly homologous to a vps mutation of yeast. VPS33A thus is a good candidate gene for a previously uncharacterized form of human HPS.

Group A rotaviruses (RVA) are the leading cause of severe diarrhea in young children worldwide. Two live-attenuated RVA vaccines, Rotarix(®) and RotaTeq(®) are recommended by World Health Organization (WHO) for routine immunization of all infants. Rotarix(®) and RotaTeq(®) vaccines have substantially reduced RVA associated mortality but occasionally have been associated with acute gastroenteritis (AGE) cases identified in vaccinees and their contacts. High-throughput assays are needed to monitor the prevalence of vaccine strains in AGE cases and emergence of new vaccine-derived strains following RVA vaccine introduction. In this study, we have developed quantitative real-time RT-PCR (qRT-PCR) assays for detection of Rotarix(®) and RotaTeq(®) vaccine components in stool samples. Real-time RT-PCR assays were designed for vaccine specific targets in the genomes of Rotarix(®) (NSP2, VP4) and RotaTeq(®) (VP6, VP3-WC3, VP3-human) and validated on sequence confirmed stool samples containing vaccine strains, wild-type RVA strains, and RVA-negative stools. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments. Rotarix(®) NSP2 and VP4 qRT-PCR assays exhibited 92-100% sensitivity, 99-100% specificity, 94-105% efficiency, and a limit of detection of 2-3 copies per reaction. RotaTeq(®) VP6, VP3-WC3, and VP3-human qRT-PCR assays displayed 100% sensitivity, 94-100% specificity, 91-102% efficiency and limits of detection of 1 copy, 2 copies, and 140 copies, respectively. These assays permit rapid identification of Rotarix(®) and RotaTeq(®) vaccine components in stool samples from clinical and surveillance studies and will be helpful in determining the frequency of vaccine strain-associated AGE.

A real-time quantitative reverse transcription-PCR (qRT-PCR) assay using the recombinant thermostable Thermus thermophilus (r Tth ) enzyme was developed to detect and quantify rotavirus A (RVA). By using r Tth polymerase, significant improvement was achieved over the existing real-time RT-PCR assays, which require denaturation of the RVA double-stranded RNA (dsRNA) prior to assay setup. Using a dsRNA transcript for segment 7, which encodes the assay target NSP3 gene, the limit of detection for the improved assay was calculated to be approximately 1 genome copy per reaction. The NSP3 qRT-PCR assay was validated using a panel of 1,906 stool samples, 23 reference RVA strains, and 14 nontarget enteric virus samples. The assay detected a diverse number of RVA genotypes and did not detect other enteric viruses, demonstrating analytical sensitivity and specificity for RVA in testing stool samples. A XenoRNA internal process control was introduced and detected in a multiplexed qRT-PCR format. Because it does not require an antecedent dsRNA denaturation step, this assay reduces the possibility of sample cross-contamination and requires less hands-on time than other published qRT-PCR protocols for RVA detection.

OBJECTIVE: Previous US evaluations have not assessed monovalent rotavirus vaccine (RV1, a G1P[8] human rotavirus strain) effectiveness, because of its later introduction (2008). Using case-control methodology, we measured the vaccine effectiveness (VE) of the 2-dose RV1 and 3-dose pentavalent vaccine (RV5) series against rotavirus disease resulting in hospital emergency department or inpatient care. METHODS: Children were eligible for enrollment if they presented to 1 of 5 hospitals (3 in Georgia, 2 in Connecticut) with diarrhea of ≤10 days’ duration during January through June 2010 or 2011, and were born after RV1 introduction. Stools were collected; immunization records were obtained from providers and state electronic immunization information system (IIS). Case-subjects (children testing rotavirus antigen-positive) were compared with 2 control groups: children testing rotavirus negative and children selected from IIS. RESULTS: Overall, 165 rotavirus-case subjects and 428 rotavirus-negative controls were enrolled. Using the rotavirus-negative controls, RV1 VE was 91% (95% confidence interval [CI] 80 to 95) and RV5 VE was 92% (CI 75 to 97) among children aged ≥8 months. The RV1 VE against G2P[4] disease was high (94%, CI 78 to 98), as was that against G1P[8] disease (89%, CI 70 to 96). RV1 effectiveness was sustained among children aged 12 through 23 months (VE 91%; CI 75 to 96). VE point estimates using IIS controls were similar to those using rotavirus-negative controls. CONCLUSIONS: RV1 and RV5 were both highly effective against severe rotavirus disease. RV1 conferred sustained protection during the first 2 years of life and demonstrated high effectiveness against G2P[4] (heterotypic) disease.

Rotaviruses are a major cause of viral gastroenteritis in children. For accurate and sensitive detection of rotavirus RNA from stool samples by reverse transcription-polymerase chain reaction (RT-PCR), the extraction process must be robust. However, some extraction methods may not remove the strong RT-PCR inhibitors known to be present in stool samples. The objective of this study was to evaluate and compare the performance of six extraction methods used commonly for extraction of rotavirus RNA from stool, which have never been formally evaluated: the MagNA Pure Compact, KingFisher Flex and NucliSENS® easyMAG® instruments, the NucliSENS® miniMAG® semi-automated system, and two manual purification kits, the QIAamp Viral RNA kit and a modified RNaid® kit. Using each method, total nucleic acid or RNA was extracted from eight rotavirus-positive stool samples with enzyme immunoassay optical density (EIA OD) values ranging from 0.176 to 3.098. Extracts prepared using the MagNA Pure Compact instrument yielded the most consistent results by qRT-PCR and conventional RT-PCR. When extracts prepared from a dilution series were extracted by the 6 methods and tested, rotavirus RNA was detected in all samples by qRT-PCR but by conventional RT-PCR testing, only the MagNA Pure Compact and KingFisher Flex extracts were positive in all cases. RT-PCR inhibitors were detected in extracts produced with the QIAamp Viral RNA Mini kit. The findings of this study should prove useful for selection of extraction methods to be incorporated into future rotavirus detection and genotyping protocols.

Global rotavirus surveillance has led to the detection of many unusual human rotavirus (HRV) genotypes. The aim of this study was to elucidate the genetic and evolutionary relationships of short fragments of all 11 gene segments of G10 HRV strains identified in West Africa through the African Rotavirus Network (ARN) system. During 1998–2004 surveillance within the ARN, we identified 5 G10 P[8] HRV strains. Fragments of all 11 gene segments of these G10 strains were sequenced. Phylogenetic and sequence analyses of each gene segment revealed high nucleotide similarities amongst the ARN strains (97–100%) except in the case of the VP1(85–96%) and NSP2 genes (87.8–99.7%) where some strains were divergent. All genes of the ARN strains were classified as Wa-like (genotype 1) with the exception of their VP7 gene of all strains (genotype G10) and the VP6 gene of a single strain, 6755/2002/ARN (DS-1 like, genotype 2). While classified as Wa-like, the NSP2 genes of four of the ARN strains occupied a distinct sub-lineage related to simian strain Tuch, while the NSP2 of strain 6755/2002/ARN and NSP5 genes of all strains were closely related to the cognate genes of both human and animal strains belonging to the Wa-like genogroup. Although these findings help to elucidate the evolution of ARN G10 strains, additional sequence studies of cognate animal rotavirus genes are needed to determine irrefutably the specific origin of those genes relative to both human and animal rotavirus strains.

In the mouse, at least 16 genes regulate vesicle trafficking to specialized lysosome-related organelles, including platelet dense granules and melanosomes. Fourteen of these genes have been identified by positional cloning. All 16 mouse mutants are models for the genetically heterogeneous human disease, Hermansky-Pudlak Syndrome (HPS). Five HPS genes encode known vesicle trafficking proteins. Nine genes are novel, are found only in higher eukaryotes and encode members of three protein complexes termed BLOCs (Biogenesis of Lysosome-related Organelles Complexes). Mutations in murine HPS genes, which encode protein co-members of BLOCs, produce essentially identical phenotypes. In addition to their well-known effects on pigmentation, platelet function and lysosome secretion, HPS genes control a wide range of physiological processes including immune recognition, neuronal functions and lung surfactant trafficking. Studies of the molecular functions of HPS proteins will reveal important details of vesicle trafficking and may lead to therapies for HPS.

Hermansky-Pudlak syndrome (HPS; MIM 203300) is a genetically heterogeneous disorder characterized by oculocutaneous albinism, prolonged bleeding and pulmonary fibrosis due to abnormal vesicle trafficking to lysosomes and related organelles, such as melanosomes and platelet dense granules. In mice, at least 16 loci are associated with HPS, including sandy (sdy; ref. 7). Here we show that the sdy mutant mouse expresses no dysbindin protein owing to a deletion in the gene Dtnbp1 (encoding dysbindin) and that mutation of the human ortholog DTNBP1 causes a novel form of HPS called HPS-7. Dysbindin is a ubiquitously expressed protein that binds to alpha- and beta-dystrobrevins, components of the dystrophin-associated protein complex (DPC) in both muscle and nonmuscle cells. We also show that dysbindin is a component of the biogenesis of lysosome-related organelles complex 1 (BLOC-1; refs. 9-11), which regulates trafficking to lysosome-related organelles and includes the proteins pallidin, muted and cappuccino, which are associated with HPS in mice. These findings show that BLOC-1 is important in producing the HPS phenotype in humans, indicate that dysbindin has a role in the biogenesis of lysosome-related organelles and identify unexpected interactions between components of DPC and BLOC-1.

Hermansky-Pudlak syndrome (HPS) is a genetically heterogeneous disease involving abnormalities of melanosomes, platelet dense granules and lysosomes. Here we have used positional candidate and transgenic rescue approaches to identify the genes mutated in ruby-eye 2 and ruby-eye mice (ru2 and ru, respectively), two 'mimic' mouse models of HPS. We also show that these genes are orthologs of the genes mutated in individuals with HPS types 5 and 6, respectively, and that their protein products directly interact. Both genes are previously unknown and are found only in higher eukaryotes, and together represent a new class of genes that have evolved in higher organisms to govern the synthesis of highly specialized lysosome-related organelles.

Hermansky-Pudlak Syndrome (HPS) is a genetically heterogeneous disease characterized by defects in trafficking of intracellular vesicles to lysosome-related organelles such as melanosomes, lung lamellar bodies and platelet dense granules. The ashen (rab27a) mouse coat color mutant maintained at Roswell Park Cancer Institute (ash-Roswell) has been proposed as an animal model of HPS based upon prolonged bleeding times, decreased platelet aggregation and greatly decreased platelet dense granule components, including serotonin and ADP. However, human patients with Rab27a deficiency (Griscelli Syndrome) and other isolates of mouse ashen mutants do not exhibit prolonged bleeding suggesting an additional mutation in a gene affecting platelets but not melanocytes in the ash-Roswell mouse. In order to identify this mutation, a backcross between ash-Roswell and the PWK control strain was analyzed. In 390 backcross progeny, deficient platelet serotonin and diluted coat color (Rab27a) phenotypes segregated independently and in the proportions expected for single gene traits. The gene for serotonin deficiency was narrowed to a 2.1Mb region of mouse chromosome 10 containing 15 genes. None of these genes have been implicated previously in platelet storage pool deficiency. These studies indicate this genomic region contains a gene which uniquely controls platelet dense granules and is a candidate gene for a new form of human HPS.